Ataxia telangiectasia mutated (ATM) is associated with radiation sensitivity, impaired cell cycle checkpoint control, and DNA double-strand break (DSB) repair. There is evidence that in response to radiation, ATM signaling is transmitted through several of the stress-activated protein kinase pathways and is involved in G1/S, S and G2/M cell cycle check-point control some of which work through P53. A direct involvement of ATM in DSB repair has not been conclusively demonstrated except that AT cells are impaired in homologous recombination (HR) and ATM directly phosphorylates key proteins involved in this type of repair. In this proposal, we hypothesize that distinct signaling pathways are triggered by nuclear and extra-nuclear events in response to DSBs generated by an endonuclease and clinically relevant doses of radiation, which affect DSB repair and, importantly, can be targeted by genetic manipulation to radiosensitize tumors. We propose to generate DSBs from an adenovirus expressing a restriction endonuclease that will induce stress responses that can be regarded as being triggered by "pure" DNA damage. The signaling responses elicited by this type of DSB will be investigated and related to ATM and P53 function, and other factors involved in HR, and to cell cycle regulation. Furthermore, we will determine qualitative and quantitative changes in DSB repair (HR and non-homologus end-joining) using an in vivo DSB repair assay based on I-Scel, a rare-cutting endonuclease from yeast, after manipulating cells with different genetic backgrounds with a variety of pharmacological and genetic reagents. We will determine whether stress responses generated from endonuclease and radiation affect DSB repair. Radiation triggers extra-nuclear-generated events that feed through growth factor receptors in addition to DNA damage. We will also determine critical signaling pathways involved in DSB repair that are influenced by growth and the cell cycle. We expect these studies to lead to a greater understanding of the signaling processes important for DSB repair that can be used to design novel genetic therapeutic strategies. We will explore the feasibility of targeting DSB repair and/or kinase cascades important for DSB repair with dominant-negative (DN) adenoviruses to radiosensitize gliomas.